Method for assaying proteolytic enzymes

ABSTRACT

A reagent for proteolytic enzyme assays has the general formula ##STR1## where RCO-- is an enzyme reactive acyl, such as an amino acid, peptide or substituted amino acid or peptide. The reagent may be hydrolysed by proteolytic enzymes and developed to form a distinctive color. The reagent may be formed by reacting RCOOH with N-hydroxysuccinimide to form the acyl N-hydroxysuccinimide ester. The ester may then be reacted to form the reagent.

BACKGROUND AND SUMMARY OF THE INVENTION

Proteolytic assays play an important role in the diagnosis ofpathological conditions, such as abnormalities in coagulation, incomplement systems and in the identification of infectous agents. Commonreagents for this purpose are paranitroanilide (PNA) derivatives ofamino acids and peptides. PNA is commonly used to measure proteolyticactivity. When contacted with enzymes present as a result of aparticular pathological condition the PNA is released The presence offree PNA is measured colorimetrically. The released PNA is a yellow dyehaving an absorption maximum of 405 nanometers.

While PNA analysis if very useful, many body fluids, such as plasma,urine and spinal fluid absorb strongly at the wave length of PNA andthus give a very high background, which interfers with analysis of PNAtests. Applicants have discovered that it is advantageous to have anenzyme analysis system which works with a substrate having an absorptionmaximum at greater than about 500 nanometers, since this range(typically a blue/green color) provides high contrast and sensitivityand is not obscured by the background color of many body fluids.Applicants' have discovered and produced a series of such reagentmaterials which are effective in proteolytic enzyme assays and which canbe read visually or electronically. Applicants' reagents can be usedboth in manual and in automatic analytical systems.

Applicants' have discovered that enzyme reactive acyl groups can becombined with a dye precursor, for example, a thizaolinone hydrazone(TH) or substituted thiazolinone hydrazone such as3-methyl-2-benzothiazolinone hydrazone (MBTH) and the like, to produce areagent which can be cleaved by proteolytic enzymes to free an indicatordye precursor, which can be developed. Applicants' reagent and systemproduce a highly sensitive, high contrast test.

It is thus an object of applicants' invention to produce a high contrastproteolytic enzyme test reagent.

It is a further object of applicants' invention to produce a testreagent which has an adsorption maximum in the blue/green range.

It is an object of applicants' invention to produce a test reagent whichutilizes an enzyme reactive acyl group combined with a dye moiety.

It is a further object of applicants' invention to provide a method ofproducing an enzyme assay substrate which will release a dye precursor.

It is a further object of applicants' invention to provide a method oftesting proteolytic enzyme activity using a reagent substrate which willrelease a dye moiety on contact with a proteolytic enzyme.

DESCRIPTION OF THE DRAWING

The figure is a graph of the hydrolysis rate of one of applicants'substrates.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

A new class of proteolytic enzyme substrates has been synthesized. Thesecompounds are ¹ N-acyl derivatives of thiazolinone hydrazone orsubstituted thiazolinone hydrazone having the formula ##STR2## in whichR₁, R₂ and R₃ may be H, aliphatic, cyclic, fused cyclic, aromatic, fusedaromatic, carboxylic, halogen, nitro, hydroxyl, amino, thio, esters andthe like dye precusor substituents. For example,3-methyl-2-benzothiazolinone hydrazone (MBTH) and substituted MBTHproduce superior blue dyes. Preferably, RCO-- is an amino acid, forexample any of the 20 common amino acids, or an amino acid derivative,including peptides and their derivatives. The new substrates may bysynthesized by the standard techniques of peptide synthesis. Applicantshave found, however, that a particularly advantageous synthesis is onewhich reacts the acyl precursor with N-hydroxysuccinimide to produce anester, followed by reacting the ester with the thilazolinone hydrazone.

The RCO-TH hydrolysis is catalyzed by proteolytic enzymes to give acarboxylic acid RCOOH and TH. The hydrolysis product TH may be detectedspectrophotometrically by oxidative coupling with aromatic amines orphenols, for example 3-dimethylaminobenzoic acid (DMAB) or equivalentoxidative coupling agents. The reaction mixtures for detection of thecolor preferably include an oxidizing agent, for example, ferricyanideor the peroxidase-hydrogen peroxide system, for color development. Thedye produced in the system with MBTH and DMAB maximally absorbs at 590nm and is intensely colored, with molar absorptivity of 57,000 M⁻¹ cm⁻¹at the typical assay conditions.

The invention may be more thoroughly understood by reference to theincluded examples which describe the synthesis of amides of the typeRCO-MBTH (Examples 1-15) and a continuous enzyme assay (Example 16). Adiscontinuous assay is illustrated in Example 17. Example 18 is adetermination of the kinetic catalytic constant (k_(c)) and bindingconstant (K_(m)) for trypsin-catalyzed hydrolysis of an RCO-MBTHsubstrate by the continuous assay method.

Suitable criteria for identification and purity of applicants'substrates per the above examples generally include: (1) production ofat least 90% of the theoretically calculated amount of blue color at 590nm in the standard color test upon cleavage by trypsin; (2) producthomogeneity or near homogeneity as indicated by partition thin-layerchromatography on silica gel in a solvent system of sec-butyl alcohol/3%aqueous ammonia (100:44 V/V) with spot detection by iodine vapor andlong-wavelength ultraviolet light; (3) agreement between thetheoretically expected and experimentally obtained proton-decoupled C-13nuclear magnetic resonance spectra in 99.9 atom %D dimethylsulfoxide-d₆15.0 at MHz. (4) amino acid analysis of hydrolysates of RCO-TH. Thefirst test, color development of tryptic digests, is limited tocompounds containing an Arg-MBTH or Lys-MBTH linkage. These amino acidresidues are required to obtain sufficient tryptic reactivity to carrythe reaction to completion with a reasonable time for the test. Otheramino acid-MBTH linkages may be split more rapidly by other enzymes, butthe above criteria are suitable as a standard.

EXAMPLE 1 ¹ N-(N-t-butoxycarbonyl)-L-phenylalanyl3-methyl-2-benzothiazolinone hydrazone)

In a 125-ml erlenmeyer flask, 35 mmol (12.7 g) of theN-hydroxysuccinimide ester of Boc-L-phenylalanin® was added to 70 mL ofacetonitrile (HPLC grade) followed by the addition of 33.4 mmol (6.0 g)of 3-methyl-2-benzothiazolinone hydrazone (MBTH). The reaction mixturewas stirred at room temperature for 24 h. The colorless precipitate wascollected by filtration washed with 10 mL cold acetonitrile and dried atreduced pressure

Boc-L-PHE-MBTH: Yield 11.8 g (83%), m.p. 182° C.

EXAMPLE 2 ¹ N-(N-benzyloxycarbonyl)-L-phenylalanyl3-methyl-2-benzothiazolinone hydrazone)

In a 125-mL erlenmeyer flask, 35 mmol (13.9 g) of theN-hydroxysuccinimide ester of Z-L-phenylalanine was added to 70 mL ofacetonitrile (HPLC grade) followed by the addition of 33.4 mmol (6.0 g)of 3-methyl-2-benzothiazolinone hydrazone. The reaction mixture wasstirred at room temperature for 24 h. The colorless precipitate wascollected by filtration, washed with 10 mL cold acetonitrile and driedat reduced pressure.

Z-L-PHE-MBTH: Yield 12.3 g (80%), m.p. 178° C.

EXAMPLE 3 ¹ N-(N-benzyloxycarbonyl)-D-phenylalanyl3-methyl-2-benzothiazolinone hydrazone)

In a 125-mL erlenmeyer flask, 35 mmol (13.9 g) of theN-hydroxysuccinimide ester of Z-D-phenylalanine was added to 70 mL ofacetonitrile (HPLC grade) followed by the addition of 33.4 mmol (6.0 g)of 3-methyl-2-benzothiazolinone hydrazone. The reaction mixture wasstirred at room temperature for 24 h. The colorless precipitate wascollected by filtration, washed with 10 mL cold acetonitrile and driedat reduced pressure.

Z-D-PHE-MBTH: Yield 11.4 g (74%), m.p. 176° C.

EXAMPLE 4 ¹ N-(N-t-butoxycarbonyl)-L-alanyl 3-methyl-2-benzothiazolinone hydrazone)

In a 125-mL erlenmeyer flask, 35 mmol (10.0 g) of theN-hydroxysuccinimide ester of Boc-L-alanine was added to 70 mL ofacetonitrile (HPLC grade) followed by the addition of 33.4 mmol (6.0 g)of 3-methyl-2-benzothiazolinone hydrazone. The reaction mixture wasstirred at room temperature for 24 h. The colorless precipitate wascollected by filtration, washed with 10 mL cold acetonitrile and driedat reduced pressure.

Boc-L-ALA-MBTH: Yield 7.8 g (67%), m.p. 181° C.

EXAMPLE 5 ¹ N-(N-t-butoxycarbonyl)-S-benzyl-L-cysteinyl3-methyl2-benzothiazolinone hydrazone)

In a 125 mL erlenmeyer flask, 35 mmol (14.3 g) of theN-hydroxysuccinimide ester of Boc-L-(Bz)cysteine was added to 70 mL ofacetonitrile (HPLC grade) followed by the addition of 33.4 mmol (6.0 g)of 3-methyl-2-benzothiazolinone hydrazone. The reaction mixture wasstirred at room temperature for 24 h. The colorless precipitate wascollected by filtration, washed with 10 mL cold acetonitrile and driedat reduced pressure.

Boc-L-(Bz)CYS-MBTH: Yield 11.3 g (72%), m.p., 144°-146° C.

EXAMPLE 6 ¹ N-(N-t-butoxycarbonyl)-L-methionyl3-methyl-2-benzothiazolinone hydrazone)

In a 125-mL erlenmeyer flask, 35 mmol (12.1 g) of theN-hydroxysuccinimide ester of Boc-L-methionine was added to 70 ml ofacetonitrile (HPLC grade) followed by the addition of 33.4 mmol (6.0 g)of 3-methyl-2-benzothiazolinone hydrazone. The reaction mixture wasstirred at room temperature for 24 h. The colorless precipitate wascollected by filtration, washed with 10 mL cold acetonitrile and driedat reduced pressure.

Boc-L-MET-MBTH: Yield 10.7 g (78%), m.p. 157° C.

EXAMPLE 7 ¹ N-(N-benzyloxycarbonyl)-L-prolyl3-methyl-2-benzothiazolinone hydrazone)

In a 125-mL erlenmeyer flask, 35 mmol (12.1 g) of theN-hydroxysuccinimide ester of Z-L-proline was added to 70 mL ofacetonitrile (HPLC grade) followed by the addition of 33.4 mmol (6.0 g)of 3-methyl-2-benzothiazolinone hydrazone. The reaction mixture wasstirred at room temperature for 24 h. The colorless precipitate wascollected by filtration, washed with 10 mL of cold acetonitrile anddried at reduced pressure.

Z-L-PRO-MBTH: Yield 7.1 g (52%), m.p. 188° C.

EXAMPLE 8 ¹ N-(N-t-butoxycarbonyl-γbenzyl-L-glutamyl3-methyl-2-benzothiazolinone hydrazone)

In a 125-mL erlenmeyer flask, 35 mmol (15.3 g) of theN-hydroxysuccinimide ester of Boc-L-(Bz)glutamic acid was added 70 mL ofacetonitrile (HPLC grade) followed by the addition of 33.4 mmol (6.0 g)of 3-methyl-2-benzothiazolinone hydrazone. The reaction mixture wasstirred at room temperature for 24 h. The colorless precipitate wascollected filtration, washed with 10 mL cold acetonitrile and dried atreduced pressure.

Boc-L-(Bz)GLU-MBTH: Yield 12.3 g (74%), m.p. 131°-133° C.

EXAMPLE 9 ¹ N-(N-t-butoxycarbonyl)glycyl 3-methyl-2-benzothiazolinonehydrazone)

In a 125-mL erlenmeyer flask, 35 mmol (9.5 g of the N-hydroxysuccinimideester of Boc-glycine was added to 70 mL of acetonitrile (HPLC grade)followed by the addition of 33.4 mmol (6.0 g) of3-methyl-2-benzothiazolinone hydrazone. The reaction mixture was stirredat room temperature for 24 h. The colorless precipitate was collected byfiltration, washed with 10 mL cold acetonitrile, and dried at reducedpressure.

Boc-GLY-MBTH: Yield 5.75 g (51%), m.p. 169°-170° C.

EXAMPLE 10 ¹ N-(N-t-butoxycarbonyl)-L-leucyl3-methyl-2-benzothiazolinone hydrazone)

In a 125-mL erlenmeyer flask, 35 mmol (11.5 g) of theN-hydroxysuccinimide ester of Boc-L-leucine was added to 70 mL ofacetonitrile (HPLC grade) followed by the addition of 33.4 mmol (6.0 g)of 3-methyl-2-benzothiazolinone hydrazone. The reaction mixture wasstirred at room temperature for 24 h. The colorless precipitate wascollected by filtration, washed with 10 mL cold acetonitrile and driedat reduced pressure.

Boc-L-LEU-MBTH: Yield 8.5 g (70%), m.p. 120° C.

EXAMPLE 11 ¹ N-(N-t-butoxycarbonyl)-O-benzyl-L-threonyl3-methyl-2-benzothiazolinone hydrazone)

In a 125-mL erlenmeyer flask, 35 mmol (14.2 g) of theN-hydroxysuccinimide ester of Boc-L-(Bz)threonine was added to 70 mL ofacetonitrile (HPLC grade) followed by the addition of 33.4 mmol (6.0 g)of 3-methyl-2-benzothiazolinone hydrazone. The reaction mixture wasstirred at room temperature for 24 h. The colorless precipitate wascollected by filtration, washed with 10 mL cold acetonitrile and driedat reduced pressure.

Boc-L-(Bz)THR-MBTH: Yield 13.5 g (86%), m.p. 122°-126° C.

EXAMPLE 12 ¹ N-(Di-N-benzyloxycarbonyl)-L-1Ysyl3-methyl-2-benzothiazolinone hydrazone)

In a 125-mL erlenmeyer flask, 35 mmol (17.9 g) of the Nhydroxysuccinimide ester of Di-Z-L-lysine was added to 70 mL ofacetonitrile (HPLC grade) followed by the addition of 33.4 mmol (6.0 g)of 3-mthyl-2-benzothiazolinone hydrazone. The reaction mixture wasstirred at room temperature for 24 h. The colorless precipitate wascollected by filtration, washed with 10 mL cold acetonitrile and driedat reduced pressure.

Di-Z-L-LYS-MBTH: Yield 17.0 g (88%), m.p. 169°-170° C.

EXAMPLE 13 ¹ N-(N-α-t-butoxycarbonyl)argininyl3-methyl-2-benzothiazolinone hydrazone)

A solution of 6.68 g (21.5 mmol) of (N-α-t-butoxycarbonyl)argininehydrochloride and 2.17 g (21.5 mmol) of N-methylmorpholine in 50 mL ofdry N,N-dimethylformamide (DMF) in a 100-mL round-bottomed flask wascooled to -10° C. in an ice-salt bath. To the stirred, cooled solutionwas added dropwise with stirring 2.93 g (21.5 mmol) of isobutylchloroformate. The reaction mixture was maintained at -10° C. for 15minutes, then 3.85 g (21.5 mmol) of 3-methyl-2-benzothiazolinone (MBTH)was added in one batch. Gas evolution and a change from colorless toamber was observed within 1-5 minutes of the addition of the MBTH, afterwhich the reaction mixture was permitted to come to room temperature for24 h. The precipitated N-methylmorpholine hydrochloride was removed byfiltration and the clear amber supernatent was added to 500 ml, ofdeionized water. The aqueous solution was brought to pH 8 by addition ofsolid sodium bicarbonate. Occasionally a color change from amber togreen was observed upon adjustment of the pH. The solution wastransferred to a 2 L separatory funnel, shaken vigorously with 800 mL ofmethylene chloride, and the yellow methylene chloride layer was removed.Upon standing at room temperature for 5 minutes to 8 h, a solid productprecipitated from the colorless aqueous layer. The product was removedby filtration and dried at reduced pressure to give a colorless or paleyellow powder as the bicarbonate salt of the product.

EXAMPLE 14 ¹ N-(N-benzoyl-L-phenylalanyl-L-valyl)-L-arginyl3-methyl-2-benzothiazolinone hydrazone)

In a 125-mL erlenmeyer flask, 1.6 mmol (0.90 g) of theN-hydroxysuccinimide ester of N-benzoyl-L-phenylalanyl-L-valine wasdissolved in 20 mL acetonitrile (HPLC grade). A solution containing 4.8mmol (0.40 g) of sodium bicarbonate in 40 mL of water was added, withstirring, followed by 1.6 mmol (0.72 g) of 1N-arginyl3-methyl-2-benzothiazolinone hydrazone-3HCl. After 24 h at roomtemperature, the colorless precipitate was collected by filtration andwashed with 10 mL of cold water and dried at reduced pressure. Thebicarbonate salt of the product was produced in 71% yield (0.82 g).

EXAMPLE 15 ¹ N-(N-t-butoxycarbonyl-L-leucylglycyl)-L-arginyl3-methyl-2-benzothiazolinone hydrazone)

In a 50-mL erlenmeyer flask, 3.9 mmol (1.5 g) of theN-hydroxysuccinimide ester of N-t-butoxycarbonyl-L-leucyl glycinedissolved in 20 mL of dry dimethylformamide (HPLC grade). With stirring,13 mL of a 5% (w/v) solution of sodium bicarbonate was added, followedby a 3.9 mmol (1.75 g) of the trihydrochloride salt of ¹ N-arginyl3-methyl-benzothiazolinone hydrazone. After 24 h at room temperature theresidual sodium bicarbonate was removed by filtration. Evaporation ofthe filtrate at reduced pressure produced the bicarbonate salt of thetitle compound as a pale yellow solid. The product was washed twice with10 mL of cold water and dried at reduced pressure: 67% yield (1.76 g).

EXAMPLE 16 Saturation kinetics forN-(N-benzyloxycarbonyl)glycyl-L-prolyl-L-arginyl3-methyl-2-benzothiazolinone hydrazone with trypsin).

A continuous assay for trypsin was performed, i.e., the oxidizing andcoupling reagents for color development were included with the substratein the reaction mixture so that color development may be monitored on acontinuous basis with time. In this example the dye precursor isdeveloped to a dye without destroying the enzymatic activity of theenzyme. The reaction was initiated by the addition of 0.1 mL of enzyme(10 μg/mL) to 0.9 mL of buffer solution containing substrate(Z-GLY-PRO-ARG-MBTH) and color developing reagents. The finalconcentrations in the cuvette were 1 μg trypsin +0.14M Tris buffer, pH7.8 +0.0131M 3-dimethylamino benzoic acid (DMAB) +0.002M potassiumferricyanide +3% dimethylformamide +substrate. Substrate concentrationsof 1.74×10⁻³ M, 5.8×10⁻⁴ M and 3.9×10⁻⁴ M were assayed in triplicate.The increases in absorbance at 590 nm were monitored continuously for 5minutes and the initial rates determined. Using the molar absorptivityof 57,000 for the chromogen produced (MBTH-DMAB), the average rate ofhydrolysis was calculated to be 4.43±0.1×10⁻⁶ mol min⁻¹.

EXAMPLE 17 Discontinuous peroxidase-coupled assay of α-chymotrypsinUsing N-(N-benzyloxvcarbonyl)-L-phenylalanyl3-methyl-2-benzothiazolinone hydrazone (Z-L-PHE-MBTH).

In this example the enzyme is permitted to hydrolyse the substrate inthe absence of oxidizer and coupling agent. Color development isinitiated by addition of oxidizer and coupling agent and the colorabsorption is measured on a spectrophotometer. An amount of substrateneeded to give a 3.2 mM solution was dissolved in 10 mLdimethylformamide. To 30 mL of 0.1M phosphate buffer, pH 8.0, 1.0 mL ofthe stock substrate solution was added. The reaction was initiated bythe addition of 1.0 mL of α-chymotrypsin stock solution (50 mg/mL in0.1M acetate buffer, pH 5.0). At t=0 and periodically over a 90 minutetime span, 1.0 mL aliquots were removed and added to a 3.0 mL colordeveloping solution containing 0.0083M 3-dimethylamino benzoic acid(DMAB) +0.1% hydrogen peroxide +10 μg horseradish peroxidase. After 2minutes, the absorbence at 590 nm was recorded. At the conditions usedthe chromogen (DMAB-MBTH) had a molar absorptivity of 65,000. FIG. 1shows the rate of hydrolysis of Z-L-PHE-MBTH, as indicated by theappearance of MBTH.

EXAMPLE 18 Determination of the Kinetic Constants for the TrypsinCatalyzed Hydrolysis of N-(N-t-butoxycarbonyl)-L-Arginyl 3-methylbenzothiazolinone hydrazone (continuous assay)).

In a one cm. cuvette were mixed 3.0 mL of 0.02M tris buffer, pH 7.8,containing 0.02M 3-dimethylamino benzoic acid (DMAB) 0.1 mL of 0.065Mpotassium ferricyanide, and 0.1 mL of substrate (BOC-ARG-MBTH). Thereaction was initiated by the addition of 1 0.1 mL of trypsin (2.33×10⁻⁴M in 0.1M pyrophosphate buffer, pH 8.3). Eight concentrations ofsubstrate, from 9.98×10⁻⁴ M to 7.8×10⁻⁶ M, were assayed in triplicate.The increase in absorption at 590 nm was monitored continuously and theinitial rates were determined. Under the conditions of the assay, amolar absorptivity of 57,000 was used to determine the concentration ofthe chromogen (MBTH-DMAB) produced by the hydrolysis of substrate. TableI contains the date used to determine the binding and kinetic constants(K_(m), k_(c)). A Lineweaver-Burk plot yielded respective values ofK_(m) and k_(c) of 1.41×10⁻³ M and 4.36×10⁻² s⁻¹. analysis by theWoolf-Augustinssen-Hofstee method gave K_(m) =1.29×10⁻³ M and k_(c)=4.28×10⁻² s⁻¹.

                  TABLE 1                                                         ______________________________________                                        I/V (min M.sup.-1)                                                                           I/S (M.sup.-1)                                                                          V/S (min.sup.-1)                                     ______________________________________                                        0.000565                 0.00177                                              0.000528       100       0.00190                                              0.000625                 0.00160                                              0.000699                 0.00287                                              0.000742       200       0.00270                                              0.000742                 0.00270                                              0.000766                 0.00523                                              0.000880       401       0.00456                                              0.000819                 0.00489                                              0.001033                 0.00776                                              0.001080       802       0.00743                                              0.001188                 0.00675                                              0.001697                 0.00945                                              0.001607       1603      0.00945                                              0.001827                 0.00877                                              0.002969                 0.01808                                              0.002969       2305      0.01080                                              0.003393                 0.00945                                              0.005056                 0.01268                                              0.005056       6413      0.01268                                              0.005280                 0.01215                                              0.009901                 0.01295                                              0.009901       12826     0.01295                                              0.009506                 0.01349                                              ______________________________________                                    

We claim:
 1. A method for detecting the presence of proteolytic enzymesin an assay sample comprising:a. contacting an assay sample with athiazolinone hydrazone compound having the formula: ##STR3## whereinRCO- is any enzyme reactive acyl constituent which, in the presence ofproteolytic enzyme, will release from the hydrazone substituent,.[.sid.]. .Iadd.said .Iaddend.acyl being selected from the groupconsisting of amino acids, .Iadd.substituted amino acids,.Iaddend.peptides, and substituted peptides; and wherein R₁ is selectedfrom the group consisting of hydrogen, aliphatic, cyclic, aromatic,carboxylic, halogen, hydroxyl, amino, and thio substituents; and whereinR₂ and R₃ are selected from the group consisting of aliphatic, cyclic,aromatic, .[.cart,oxylic.]. .Iadd.carboxylic.Iaddend., halogen, nitro,hydroxyl, amino, thio, fused cyclic, and fused aromatic; b. addingoxidative coupling agents and oxidizing agents for the thiazolinone todevelop blue-green chromogens; and c. monitoring the increase inabsorbance at 590 nanometers; whereby the color develops while retainingthe enzymatic activity of the proteolytic enzyme and the concentrationof the chromogens is directly proportional to the degree of enzymaticactivity.
 2. The method of claim 1 wherein the assay method is conductedcontinuously by having the oxidative coupling agents and the oxidizingagent present in the assay with the compound of Step (a) and monitoringthe absorbance continuously with time.
 3. The method of claim 1 whereinthe assay method is conducted discontinuously by periodically removingaliquots of the assay at Step (a), adding the oxidizing agents andoxidative coupling agents of Step (b) to each aliquot and recording itsabsorbance at that period of time.
 4. The method of claim 1 wherein theconstituent attached to the enzyme reactive acyl is MBTH.
 5. The methodof claim 1 wherein R₂ and R₃ are joined in either a fused cyclic orfused aromatic ring.